Reproductive strategies in loggerhead sea turtle Caretta caretta: polyandry and polygyny in a Southwest Atlantic rookery

Introduction

Sea turtles play a vital role in global biodiversity maintenance. While some populations can grow significantly, few remain unaffected by human activities (Marcovaldi, dos Santos & Sales, 2011). Observing sea turtles is difficult since these animals are both littoral and pelagic (Moore & Ball, 2002). Consequently, many aspects of their biology and behavior, particularly those of males, remain unclear. Males stay in the ocean, hindering observations, whereas females return to their natal regions to nest, providing ample research opportunities (Bowen et al., 1993). Both sexes migrate to mating areas before nesting, with males contributing to gene flow and female natal philopatry, shaping regional genetic structure (Bowen et al., 1993; FitzSimmons et al., 1997; Reid et al., 2019). However, most studies focus on females using nest counts and mark-recapture data (Casale et al., 2023; Lasala et al., 2023), leaving male behavior underexplored.

The loggerhead sea turtle (Caretta caretta) is listed as “Vulnerable” on both the Brazilian Red List (Brasil, 2022) and the IUCN Red List (Casale & Tucker, 2017). Brazil hosts one of the largest remaining loggerhead nesting populations in the world, second only to the super-aggregations found in Oman, and eastern Florida-USA (Marcovaldi & Chaloupka, 2007). In Brazil, loggerhead nesting sites are primarily located along the northeast (Sergipe and Bahia) and southeast coast (Espírito Santo and Rio de Janeiro) (Marcovaldi, dos Santos & Sales, 2011; Marcovaldi et al., 2016). These populations are typically defined by the geographic locations of these key nesting sites along the coast.

In Espírito Santo, long-term nesting female loggerheads exhibit strong nest site fidelity, meaning they repeatedly return to the same nesting sites (Barreto et al., 2019). This behavior is a well-documented characteristic of the species (Miller, 1997) and provides a reliable opportunity for researchers to study and monitor these turtles in a consistent environment. Espírito Santo, therefore, serves as an ideal location for conducting long-term studies on loggerhead nesting behavior, population dynamics, and conservation efforts. The high fidelity to nesting sites not only facilitates the sampling of individuals but also enhances the effectiveness of conservation programs aimed at protecting these critical habitats.

Loggerheads reach sexual maturity between 25 and 35 years old (Chaloupka & Limpus, 1997; Chaloupka & Musick, 1997). Hormone analysis shows males can breed successively within a breeding season and potentially in consecutive years (Wibbels et al., 1990), and satellite tracking data confirms they stay close to breeding grounds (Hays, Mazaris & Schofield, 2014). Males likely mate more frequently than females due to lower reproductive investment. Post-mating, males return to foraging grounds, while females travel to nesting beaches (Bowen et al., 2004), laying up to seven clutches per season and return every 2 to 3 years (Dodd, 1988). In Brazil, however, the northern Brazilian loggerheads lay, on average, 3.1 clutches per season and return every 2.4 years (Marcovaldi et al., 2010).

The sex of sea turtles is determined by the temperature of their nests, otherwise known as temperature-dependent sex determination (TSD) (Montero et al., 2018). Simply put, warmer nest temperatures tend to produce more females, while cooler temperatures result in more male hatchlings (Marcovaldi, Godfrey & Mrosovsky, 1997). In addition, other factors include moisture level (Lolavar & Wyneken, 2015; Montero et al., 2018). This temperature-dependent mechanism is very important to understand the development and adaptation of sea turtle populations, as it directly influences the sex ratio in a population.

The pivotal temperature for a 1:1 sex ratio in loggerheads is around 29.3 °C (Mrosovsky et al., 2002), with a similar value of 29.2 °C recorded in Bahia, Brazil (Marcovaldi, Godfrey & Mrosovsky, 1997). In the southern regions, a near-balanced sex ratio of 53% female was found in Espírito Santo (ES) and Rio de Janeiro (RJ), while a strong female bias of 94% was observed in the northern regions, Sergipe (SE) and Bahia (BA) (Marcovaldi et al., 2016).

Global temperature increases could skew these ratios, impacting adult sex ratios (Mrosovsky & Provancha, 1992; Lolavar & Wyneken, 2015). Increased male breeding frequency may balance female-skewed hatchling ratios, producing a more equitable operational sex ratio (Hays et al., 2010; Wright et al., 2012; Hays, Mazaris & Schofield, 2014). If males cannot be censused, breeding sex ratio (BSR) can be estimated through paternity testing.

Studies show male reproductive patterns significantly affect long-term population persistence (Mitchell & Janzen, 2010; Hays, Mazaris & Schofield, 2014). Female sea turtles often mate with multiple males, resulting in nests with multiple paternal contributions (Lee et al., 2018). The extent of multiple mating remains uncertain (Moore & Ball, 2002), but females can store sperm in the oviduct after mating (Uller & Olsson, 2008) for at least 3 months (Sakaoka et al., 2013; Lasala, Hughes & Wyneken, 2020). Polyandry, as evidenced by multiple paternity (MP), is a common mating strategy observed across several sea turtle species. This strategy has been documented in species such as the green turtle (Chelonia mydas) (Turkozan et al., 2019), hawksbill (Eretmochelys imbricata) (González-Garza et al., 2015), leatherback (Dermochelys coriacea) (Stewart & Dutton, 2014), olive ridley (Lepidochelys olivacea) (Duran et al., 2015), Kemp’s ridley (Lepidochelys kempii) (Kichler et al., 1999), and flatback (Natator depressus) (Theissinger et al., 2009).

Multiple paternity in loggerheads has been well-documented, with females typically mating with several males and consequently laying clutches that carry varied paternal contributions. In the United States, specifically in Florida, levels of polyandry range from 22% to 70% (Bollmer et al., 1999; Moore & Ball, 2002; Lasala, Hughes & Wyneken, 2018, 2020), while in Georgia, this figure reaches 78% (Lasala et al., 2013). In Australia, the levels of multiple paternity vary between 48% (Tedeschi et al., 2015) to 65.5% (Howe et al., 2018). In Turkey, the percentage is 70% (Sari, Koseler & Kaska, 2017). Greece, on the other hand, reports the highest level of multiple paternity ever recorded for loggerheads, at 95% (Zbinden et al., 2007). In Japan, two studies reported levels of 42.9% (Sakaoka et al., 2011) and 27.3% (Sakaoka et al., 2013), respectively; however, these studies were conducted on captive turtles. These findings highlight the complex mating system of loggerhead turtles and underscore the importance of incorporating multiple paternity into conservation strategies.

Improving our understanding of multiple paternity and BSR is critical in predicting the long-term persistence of the Brazilian loggerhead populations. Therefore, this study is a crucial step in the conservation of sea turtles. We therefore provide some basic knowledge on the extent of multiple paternity in loggerhead nests on the Atlantic coast in Espírito Santo, Brazil, together with an estimate of the breeding sex ratio for this population. This research not only fills gaps in our knowledge but also provides a base for future studies that will ensure the survival and genetic health of loggerhead turtles in Brazil and beyond. More specifically, the main objectives of this study were to test for multiple paternity in loggerhead nests along the Atlantic coast in a rookery of Espírito Santo, Brazil, and to estimate the breeding sex ratio for this population. In addition, this study aims to investigate whether there is a relationship between female size and the number of males contributing to the nest. Understanding this correlation could provide insights into how phenotypic traits such as female size may influence reproductive strategies and the multiple paternity.

Materials and Methods

Study area

The sampling of nesting females took place along 13 km of Povoação Beach in the Linhares district, located in the northern part of the state of Espírito Santo, Brazil (Fig. 1). Povoação Beach is part of the extensive 411-kilometer-long coastline of Espírito Santo, which stretches from the border with Bahia to the north and extends southward to the border with Rio de Janeiro, along the South Atlantic Ocean (Albino, Contti Neto & Oliveira, 2016). Povoação Beach is characterized as an exposed siliciclastic beach, associated with sandy terraces (Albino, Contti Neto & Oliveira, 2016), located in the deltaic plain of the Doce River, where it receives significant riverine inputs (Dominguez, da Bittencourt & Martin, 1981) (Fig. 1). This beach, along with Praia de Comboios, is one of the two most important long-term nesting beaches in Espírito Santo and is crucial for sea turtle conservation efforts (Marcovaldi & Chaloupka, 2007).

Results

A total of 66 females corresponding to 69 natural nests were monitored. Due to storms and predation during the incubation period, many nests were lost. A total of 1,109 hatchlings from 43 nests attributed to 42 females were initially analyzed. These nests included 17 from the 2017/18 season, 11 from the 2018/19 season, and 15 from the 2019/20 season (Fig. 2; for more details, see Table S1). However, only 534 hatchlings (48.15%) were successfully genotyped, as some samples, particularly those from residual yolks, were degraded and did not yield reliable results (Fig. 2; Table S1). Despite nearly half of the hatchlings being successfully genotyped, the number of nests and females per season remained consistent. Notably, female SMV141 nested in two different reproductive seasons (2017/18 and 2019/20), which accounts for 42 females but 43 nests. The number of hatchlings analyzed per nest ranged from 3 to 20, with an average of 12.41 hatchlings per nest.

The number of alleles per locus ranged from 10 to 13 for nesting females (N = 42) (Table 1). There was no evidence of deviation from the Hardy-Weinberg equilibrium for any loci after the Bonferroni correction. The expected heterozygosity (He) by locus for the female dataset ranged from 0.781 to 0.848, while the observed heterozygosity (Ho) by locus ranged from 0.818 to 0.932 (Table 1). There was no evidence of null alleles. The PI using the four loci was 3.2 × 10⁻⁶, and the PE found was 99.10%. The genotyping error rate was zero for the four loci used in this study.

In total, 88 distinct males were identified contributing to the hatchlings across all seasons, with 38 males in 2017/18, 24 in 2018/19, and 26 in 2019/20 (Figs. 2 and 3; Table S2). The total number of males does not represent a simple sum since some males contributed to multiple nests, either within the same season or across different seasons. For example, ♂3 contributed to both the 2017/18 and 2019/20 seasons (Figs. 2 and 3; Table S2). Polyandry, or multiple paternity, was observed in 31 out of 43 nests (72.09%), with the number of males per nest ranging from one to six, and an average of 2.04 males per nest. The Pearson’s correlation analysis between the number of genotyped hatchlings per nest and the number of inferred males revealed a significant negative correlation (p-value = 0.007 and ρ = −0.401).

Twelve males were observed to contribute to more than one nest, either within a single season or across multiple seasons (Figs. 2 and 3; Table S2). Among these, seven males displayed polygyny by fertilizing multiple females within the same season. Specifically, six of these polygynous males were active during the 2017/18 season (♂1, ♂3, ♂8, ♂21, ♂22, and ♂28), while one male was from the 2018/19 season (♂25). These males contributed to as many as three different nests within the same season (Fig. 3; Table S2). Moreover, some of these males also contributed across different seasons. For example, ♂3, ♂8, and ♂21 fertilized two nests in the 2017/18 season and another in the 2019/20 season; ♂7, ♂11 and ♂35 fertilized one nest in 2017/18 and another in 2019/20; ♂17 fertilized one nest in 2017/18 and another in 2018/19; ♂25 fertilized one nest in 2017/18 and two in 2018/19; and ♂41 fertilized one nest in 2018/19 and another in 2019/20.

Among the twelve males that fertilized more than one nest, five (♂7, ♂11, ♂17, ♂35, and ♂41) were involved in nests across multiple seasons without displaying polygyny (Figs. 2 and 3; Table S2). The polygyny rate, calculated from the total of 88 males and the seven events of polygyny, was 7.95%.

Furthermore, eight males (20.5%) from the 2017/18 season continued to contribute to fertilization in subsequent seasons. Two males returned in the 2018/19 season (♂17 and ♂25), while six males returned in the 2019/20 season (♂3, ♂7, ♂8, ♂11, ♂21 and ♂35) (Figs. 2 and 3; Table S2). This pattern suggests a degree of site fidelity and indicates a stable breeding population within the aggregation. The overall breeding sex ratio for this nesting aggregation was approximately 1 female for every 2.09 males, or 42 females to 88 males.

In examining the relationship between female size and the number of males, the data for CCL shows a slight upward trend as the number of males increases (Fig. 4). However, there is significant data variability, particularly in the groups with two and three males. The statistical analysis reveals a p-value of 0.716 and a ρ of 0.058, indicating that this trend is not statistically significant, suggesting that the apparent increase may be due to random variation rather than a true underlying effect.

Similarly, the CCW data exhibit no clear trend in relation to the number of males (Fig. 4). The distribution of the data points does not indicate a consistent pattern, with the p-value of 0.622 and ρ of 0.077 further supporting the lack of a significant correlation. This suggests that the width of the carapace does not meaningfully change with varying numbers of males.

The CA shows only modest variation across different numbers of males, with a slight increase observed in groups with five and six males (Fig. 4). However, this variation is not substantial, as indicated by the p-value of 0.645 and ρ of 0.074, which suggest that there is no significant correlation. The data imply that, much like with CCL and CCW, any observed differences in the number of males are likely due to random fluctuations rather than a direct effect of the size of females.

The results of the GLM analysis for CCL and CCW did not reveal a significant relationship between these size metrics and the number of males per nest. The GLM for CCL yielded an estimate of 0.589, Standard Error (SE) = 2.768, and p-value = 0.831, and for CCW, an estimate of 1.020, SE = 3.2444, p-value = 0.753. These results further confirm the lack of significant influence of these metrics on the number of contributing males. Similarly, the GLM analysis for CA showed no significant effect on the number of males, with an estimate of 1.373, SE = 1.429 and p-value = 0.3369. Although the intercept for CA was significant (Intercept = −2.598, SE = 1.016, p-value = 0.0106), this result suggests that carapace area does not meaningfully explain the variation in the number of fathers per nest.

Discussion

This study is the first to assess multiple paternity in loggerhead turtles in Brazil using nuclear DNA markers. Additionally, this is the first study to report instances of polygyny in loggerhead turtles. Similar high rates of polygyny have been observed in other sea turtle species, such as hawksbill turtles in El Salvador, where 31.8% of the males sired nests from multiple females (Gaos et al., 2018), and in Mediterranean green turtles in Turkey, with a polygyny rate of 4.4% (Turkozan et al., 2019). Factors that may facilitate polygyny include a more balanced primary sex ratio (Marcovaldi et al., 2016) and reproductive philopatry (FitzSimmons et al., 1997). In our study, analyzing three nesting seasons provided a broader understanding of polygyny dynamics by accounting for annual variability in the population.

In highly endangered species, the difficulty in finding mates due to a low number of males could lead to an increase in polygyny events (Crim et al., 2002). This behavior may also suggest that mating occurs closer to nesting beaches, particularly when the species’ range is limited (Natoli et al., 2017). Some studies propose that polygyny might be a consequence of a female-biased sex ratio (Crim et al., 2002). However, it remains unclear whether polygynous mating strategies can effectively counterbalance the potential impacts of feminization and the reduced availability of males (Gaos et al., 2018). Further research is needed to determine how these mating systems will adapt to ongoing environmental changes and what implications this will have for the conservation of these iconic marine species. These findings can also benefit captive breeding programs by helping to maintain genetic diversity and improve breeding success in controlled environments (Maggeni & Feeney, 2020).

Historically, research on sea turtles has predominantly concentrated on studying females, particularly during their time on the beach, resulting in a significant gap in our understanding of males (Bjorndal, 1999). This bias in research has left male’s behavior largely underexplored. When studies on male turtles do occur, they are seldom conducted through systematic, active searches. Instead, they tend to rely on incidental captures or data collected from stranded individuals (Casale et al., 2005; James, Eckert & Myers, 2005; Kawazu et al., 2014). While these methods can yield valuable insights, they are inherently limited by their reliance on chance encounters and the logistical constraints that make them impractical for many researchers. While informative, these methods are not feasible for many researchers and often rely on random chance. In contrast, the method of multiple paternity, adopted here to assess successful male contributions, is both effective and informative, offering a more reliable approach to studying male sea turtles.

The PI (3.2 × 10⁻⁶) and the PE (99.10%) obtained in this study are comparable to previously published studies on loggerhead populations and are consistent with the proposed reliability values for these indices in other taxa, as emphasized by Waits, Luikart & Taberlet (2001). Three recent articles in North America, reported similar PI and PE values: Georgia—5 × 10⁻¹³ and 99.9%, respectively; SW Florida 7.3 × 10⁻¹³ and 100%; and NW Florida: <9 × 10⁻⁸ (Lasala et al., 2013; Lasala, Hughes & Wyneken, 2018; Silver-Gorges et al., 2024). These three studies used between 5–16 microsatellites, but still had similar probability results. The power of these analyses depend on the number of alleles per locus and their frequency distribution in the population. A higher number of alleles and a balanced distribution increase the ability to distinguish between individuals, which in turn improves the PI and PE values. This suggests that the genetic markers used in this study, despite being fewer in number (four microsatellites), provide a level of resolution that is sufficient for distinguishing individual genotypes within the population (Table 1). Our high PE value further supports the robustness of our methodology, indicating a high probability of correctly excluding non-parental individuals from paternity, thereby reducing the likelihood of false positives in identifying paternity events.

The reliable detection of multiple paternity with only four microsatellites is particularly valuable for studies with limited samples, enabling more cost-effective studies of genetic diversity and reproductive behavior in other loggerhead populations and, potentially, in other species where similar challenges exist. While future research could explore the integration of additional markers or next-generation sequencing techniques to further enhance the resolution and accuracy of paternity analysis, this study provides a strong foundation for using the minimum number of microsatellites in genetic monitoring and conservation efforts.

Loggerhead turtles in Espírito Santo may exhibit higher levels of promiscuity compared to other breeding populations globally, potentially influenced by specific characteristics of the Brazilian population. This conclusion is supported by the unique mating behaviors observed, including the first documentation of polygyny in this species, suggesting a breeding strategy that differs from those seen elsewhere. This distinct pattern of mating behavior could enhance genetic diversity and reproductive success, highlighting the potential for increased promiscuity among both sexes in this population.

Alongside these observations on mating behavior, we also examined the influence of the number of genotyped hatchlings on the detection of multiple paternity. Many nests with eight to 15 genotyped hatchlings showed contributions from two to three males. This detection pattern may result from random sampling of smaller nests, or it could be that nests with lower reproductive success are hiding the true number of male contributions. While significant, the variability in the data suggests that sperm competition or fertilization success, may also play a role. Further research is needed to understand the biological and ecological drivers of this pattern.

Sperm storage, which allows females to ensure fertilization of nests throughout the nesting season (Uller & Olsson, 2008; Sakaoka et al., 2013; Lasala, Hughes & Wyneken, 2020), further contributes to this reproductive flexibility. While females can be selective in their mate choice (Kokko & Mappes, 2013), the observed contributions from multiple males across various females’ nests raise intriguing questions. This could suggest that either (a) there are fewer males available in this population, limiting female choice, or (b) this behavior represents a distinctive reproductive strategy unique to the region, reflecting adaptations to local ecological conditions.

A study analyzing mtDNA haplotypes revealed that the nesting aggregation of loggerhead turtles in Espírito Santo is genetically distinct from other rookeries in Brazil (Shamblin et al., 2014). The high rate of multiple paternity observed in this region may be attributed to increased male breeding periodicity (Hays, Mazaris & Schofield, 2014). When males and females converge in the same breeding area, the density of individuals can be up to a hundred times greater than when they move independently, potentially linking the incidence of multiple paternity to turtle density (Lee et al., 2018). This elevated occurrence of multiple paternity suggests that such behavior may not confer a clear evolutionary advantage but would rather be a byproduct of increased encounters between males and females. When comparing this with other loggerhead rookeries, we observe variable rates of multiple paternity. For example, Greece has a high rate of 93.3% (Zbinden et al., 2007), likely due to the restricted movement and high density of turtles in that area (Lee et al., 2018). In contrast, rookeries in Florida, such as Melbourne Beach, show a much lower rate of 31% (Moore & Ball, 2002), possibly due to larger foraging areas and lower densities. Similarly, Wassaw Island, Georgia, has a higher multiple paternity rate of 75% (Lasala et al., 2013), reflecting intermediate density conditions and movement patterns. Additionally, more monogamous females could be influenced by phenotypic traits or the timing of nesting, as females nesting early or late in the season may have fewer mating opportunities.

Moreover, rising evidence indicates a female-skewed hatchling sex ratio in sea turtle nesting populations (Hays, Mazaris & Schofield, 2014). In Espírito Santo, the estimated hatchling sex ratio was 53% females to 47% males (Marcovaldi et al., 2016), which is much closer to a balanced 50/50 ratio compared to other Brazilian nesting populations. For instance, the northern nesting aggregation, including Sergipe and Bahia, reported a ratio of 94% females (Marcovaldi et al., 2016). Interestingly, both green and loggerhead turtles show a high incidence of multiple paternity even in environments with biased hatchling sex ratios (e.g., 80% females) (Hays et al., 2023).

In summary, this suggests that operational sex ratios could be more balanced due to specific male mating behaviors (Hays, Mazaris & Schofield, 2014). Some studies propose that male behavior, such as frequent breeding, visiting multiple rookeries (Wright et al., 2012), and engaging in polygyny (Bell et al., 2010) can sustain sea turtle fertility even at low population sizes. The breeding sex ratio (BSR) for Espírito Santo found in this study, 2.09 males per female, aligns with other population genetic data. Loggerhead population structure is driven by female natal nesting fidelity, coupled by male mediated gene flow (Bowen et al., 2005). This BSR is consistent with regions that display female-biased primary sex ratios, which may be compensated by more male-biased operational sex ratios (Hays et al., 2010). These findings support the notion that the unique mating dynamics and genetic characteristics of loggerhead turtles in Espírito Santo are shaped by local ecological conditions, further underscoring the complexity of reproductive strategies in this population.

Studies on the reproductive behavior of sea turtles have demonstrated that polyandry is a common mating strategy (Wright et al., 2012; Lasala, Hughes & Wyneken, 2018; Lee et al., 2018). The multiple paternity rate of 72.09% found in this study aligns well with global patterns observed in other research (Fig. 5; Table S3) (Bollmer et al., 1999; Moore & Ball, 2002; Zbinden et al., 2007; Lasala et al., 2013; Tedeschi et al., 2015; Sari, Koseler & Kaska, 2017; Howe et al., 2018; Lasala, Hughes & Wyneken, 2018, 2020). Figure 5 shows significant variation in multiple paternity rates across different geographic locations, ranging from 15.8% in northwest Florida (Silver-Gorges et al., 2024) to 95% in Greece (Zbinden et al., 2007). Additionally, the average number of fathers per nest also varies, with values from 1.40 in Florida (Moore & Ball, 2002) to 3.50 in Greece (Zbinden et al., 2007) (Fig. 5; Table S3), further highlighting the diverse reproductive strategies among loggerhead turtles worldwide. The maximum number of fathers per nest observed in this study was six. This is comparable to other sea turtle species, such as leatherback, with up to three fathers (Figgener et al., 2016), green turtles, where up to ten fathers have been reported (Turkozan et al., 2019), hawksbill turtles, with up to three fathers (González-Garza et al., 2015), Kemp’s ridley turtles, with three fathers (Kichler et al., 1999), olive ridley turtles, with up to four fathers (Jensen et al., 2006), and flatback turtles, also with up to four fathers (Theissinger et al., 2009).

The variability in mating behavior may be attributed to the loggerhead’s extensive migratory distribution (Dodd, 1988), which facilitates opportunistic mating between feeding areas and migratory corridors (Lasala, Hughes & Wyneken, 2018). As global temperatures continue to rise, leading to skewed sex ratios among hatchling and juvenile populations (Jensen et al., 2018), the prevalence of multiple paternity in sea turtle clutches suggests that adult male turtles are not constrained in these breeding populations (Hays, Shimada & Schofield, 2022). Understanding these patterns is crucial for developing effective conservation strategies, especially as climate change continues to impact sea turtle populations worldwide. Future studies should aim to explore the implications of these reproductive strategies on genetic diversity and population resilience in the face of environmental changes.

For female sea turtles, multiple mating offers significant evolutionary advantages, such as increasing the likelihood of successful fertilization and enhancing the genetic diversity of their hatchlings (Andersson, 1994; Pearse & Avise, 2001; Calsbeek et al., 2007). Increased genetic diversity is especially crucial in small or recovering populations (Balazs & Chaloupka, 2004). A broad gene pool can bolster the population’s resilience against evolutionary changes and reduce the risk of inbreeding, which can lead to an extinction vortex (Frankham, Ballou & Briscoe, 2010). In the context of a warming climate, maintaining and enhancing genetic diversity through multiple paternity becomes an important evolutionary strategy.

For instance, for a small nesting population in Turkey, the high frequency of MP might be driven by population density, which increases the chances of females encountering multiple males and thus the likelihood of multiple mating events (Sari, Koseler & Kaska, 2017). However, the underlying causes of MP are complex and can vary significantly depending on geographic location, environmental conditions, and individual characteristics of the turtles.

The complexity of mating behaviors in sea turtles is evident in the way females may engage in additional mating as a strategy to avoid the high costs associated with resisting male attempts (Lee et al., 2018); however, in some species, females actively choose their mates (Lee & Hays, 2004). In Georgia, larger females significantly had more successful male contributions (Lasala et al., 2013), but in Florida, larger, presumably older, females tended to exhibit greater mate choice, resulting in a lower incidence of MP compared to younger, less experienced females (Lasala, Hughes & Wyneken, 2020). Further, in Turkey, there were no significant connections between female size and MP frequency found (Sari, Koseler & Kaska, 2017). Female mate choice may matter by nesting population. Moreover, the assumption that larger females are necessarily older is not fully supported. Phillips et al. (2021) demonstrated that sea turtles grow very slowly after reaching maturity, meaning size is not always a reliable indicator of age. Larger females likely matured close to their current size, with factors like juvenile growth rates and resource availability being key influences. Therefore, the relationship between size, age, and experience should be reconsidered in discussions of mate choice and MP in sea turtles. Additionally, cryptic female choice (CFC) could be another important mechanism influencing mating outcomes. CFC refers to a female’s ability to influence paternity after mating by selectively using or discarding sperm through physiological or biochemical processes (Firman et al., 2017). This mechanism may allow females to exert postmating control over mate selection, potentially explaining why some females show reduced rates of multiple paternity despite engaging in multiple matings.

The data presented by Sari, Koseler & Kaska (2017), along with our results (Fig. 4), indicate a high degree of multiple paternity regardless of female size. This finding supports the idea that factors such as population density and local environmental conditions may play a more significant role in determining the incidence of MP than female size alone.

The GLM analysis presented here further supports these findings. None of the size metrics showed a significant relationship with the number of males contributing to the nests. Although CA was significant, this does not suggest that carapace size fully explains the variation in the number of fathers per nest. These findings confirm that female body size is unlikely to be a key factor influencing multiple paternity in C. caretta. Instead, our data suggest that MP in C. caretta may be driven by a complex interplay of factors, including female experience, population dynamics, and potentially even the mating strategies employed by the males.

Despite concerns that climate change, particularly the warming of nesting beaches, could lead to a reduction in the number of male sea turtles, thereby limiting female mating opportunities, this may not necessarily lead to decreased genetic diversity. Males are likely capable of breeding annually, and evidence from this study suggests that males can mate with multiple females within a single breeding season. This behavior indicates that MP in sea turtles can be understood as a product of the interaction between individual behaviors, such as mate choice and mating frequency, and broader population characteristics, like density and the availability of males. These factors together help maintain a balanced BSR, even in the face of environmental challenges.

Conclusions

This research establishes a foundation for future comparative studies on the multiple paternity of loggerhead turtles in Brazil. While multiple paternity is well-documented in sea turtles, this study breaks new ground by presenting the first global evidence of polygyny in loggerheads and the first record of polyandry in this species within Brazil. Our findings highlight that some males contribute to multiple nests within and across breeding seasons, offering valuable insights into male reproductive strategies.

Future studies in Brazil should build on this work by using the same genetic markers to compare male genotypes across different nesting populations, such as those in Rio de Janeiro, Bahia, and Sergipe. This approach could clarify male gene flow between seasons and regions and provide deeper insights into reproductive population sex ratios. Expanding this research to other Brazilian sub-populations will also help estimate adult sex ratios and assess the impact of female-biased hatchling production on the Brazilian coast.

Given the significant role of multiple mating in influencing effective population size and genetic diversity, continued research on this phenomenon is crucial. Such studies will be vital for developing informed management and conservation strategies, particularly in the context of future global warming scenarios.

Supplemental Information